title: The role of metal binding and phosphorylation domains in the regulation of cisplatin-induced trafficking of ATP7B.
abstract: The copper (Cu) exporter ATP7B mediates cellular resistance to cisplatin (cDDP) by increasing drug efflux. ATP7B binds and sequesters cDDP in into secretory vesicles. Upon cDDP exposure ATP7B traffics from the trans-Golgi network (TGN) to the periphery of the cell in a manner that requires the cysteine residues in its metal binding domains (MBD). To elucidate the role of the various domains of ATP7B in its cDDP-induced trafficking we expressed a series of mCherry-tagged variants of ATP7B in HEK293T cells and analyzed their subcellular localization in basal media and after a 1 h exposure to 30 μM cDDP. The wild type ATP7B and a variant in which the cysteines in the CXXC motifs of MBD 1-5 were converted to serines trafficked out of the trans-Golgi (TGN) when exposed to cDDP. Conversion of the cysteines in all 6 of the CXXC motifs to serines, or in only the sixth MBD, rendered ATP7B incapable of trafficking on exposure to cDDP. Truncation of MBD1-5 or MBD1-6 resulted in the loss of TGN localization. Addition of the first 63 amino acids of ATP7B to these variants restored TGN localization to a great extent and enabled the MBD1-5 variant to undergo cDDP-induced trafficking. A variant of ATP7B in which the aspartate 1027 residue in the phosphorylation domain was converted to glutamine localized to the TGN but was incapable of cDDP-induced trafficking. These results demonstrate that the CXXC motif in the sixth MBD and the catalytic activity of ATP7B are required for cDDP-induced trafficking as they are for Cu-induced redistribution of ATP7B; this provides further evidence that cDDP mimics Cu with respect to the molecular mechanisms by they control the subcellular distribution of ATP7B.
authors.affiliation: Moores Cancer Center, University of California, San Diego, 3855 Health Sciences Drive, La Jolla, CA 92093-0819, USA.
authors.forename: Roohangiz
authors.initials: R
authors.lastname: Safaei
authors.forename: Preston L
authors.initials: PL
authors.lastname: Adams
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Mathews
authors.forename: Gerald
authors.initials: G
authors.lastname: Manorek
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Howell
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry*; D020816:Amino Acid Motifs; D001224:Aspartic Acid; D002384:Catalysis; D027682:Cation Transport Proteins / Q000737:chemistry*; D002945:Cisplatin / Q000737:chemistry*; D003300:Copper / Q000737:chemistry; D000073840:Copper-Transporting ATPases; D005978:Glutathione / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008670:Metals / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D021601:trans-Golgi Network / Q000737:chemistry
chemical_list: D027682:Cation Transport Proteins; D008670:Metals; D001224:Aspartic Acid; D003300:Copper; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases; D005978:Glutathione; D002945:Cisplatin